The development of an organism and ultimate function of any given cell in that organism depends on the particular set of genes being expressed (e.g., transcribed and translated) in the cell. Since virtually all the genes in the human genome have now been sequenced, the challenge now is to understand the molecular mechanisms that allow these genes to be selectively expressed.
In vertebrates, DNA methylation of CpG dinucleotides has long been identified as an important mechanism of development. DNA methylation is required for normal development (Ohki et al (1999) EMBO J 18:6653-6661; Okano et al. (1999) Cell 99:247-257); is correlated with genomic imprinting (Ashburner (1972) Results Probl Cell Differ 4:101-151; Grunstein et al. (1997) Nature 389:349-352) and is involved in X-chromosome inactivation (Heard et al. (1997) Annual Rev Genet 31:571-610). A large body of evidence indicates that cytosine methylation leads to the assembly of a specialized, heritable, repressive chromatin architecture through the recruitment of histone deacetylases (Bird and Wolffe (1999) Cell 99:451-454; Siegfried et al. (1997) Curr Biol 7:R305-307). However, the precise role of DNA methylation in tissue specific regulation of non-imprinted genes remains contentious (Bird (1997) Trends Genet 13:469-472).
Thus, DNA methylation appears to be critical in vertebrate development, which relies upon the imposition of progressively more stable states of transcriptional repression (Steinbach et al. (1997) Nature 389:395-399; Mannervik et al. (1999) Science 284:606-609). Further, DNA methylation may play a role in partitioning the genome, and the chromosomal infrastructure within which it is packaged, into active and inactive intranuclear compartments (Bird et al. (1995) Trend Genet. 11:94-99). For example, mouse primordial germ cells, embryonic stem cells and the cells of the blastocyst can progress through the cell cycle and divide without detectable DNA methylation (Lei et al. (1996) Development 122:3195-3205). Once differentiation begins, however, DNA methylation becomes essential for individual cell viability (Li et al. (1992) Cell 69:915-926; Okano et al. (1999) Cell 99:247-257).
DNA methylation has also been implicated in clinical disease states. Parasitic DNA, e.g., retrotransposons, retrovirus genomes, lentivirus genomes, L1 elements and Alu elements are known to be CpG rich. It has been proposed that DNA methylation may have arisen as a genome-defense system to silence expression of these parasitic elements and limit their spread through the genome (Yoder et al. (1997) Trend Genet. 13:335-340; Colot et al. (1999) Bio Essays 21:402-411). Additionally, several genetic diseases have been described that cause methylation defects, including the ICF syndrome (Xu et al. (1999) Nature 402:187-189), Rett syndrome (Amir et al. (1999) Nature Genet. 23:185-188) and fragile X syndrome (Oberle et al. (1991) Science 252:1711-1714).
Cellular DNA methylation patterns seem to be established by a complex interplay of at least three independent DNA methyltransferases: DNMT1, DNMT3A and DNMT3B (Kaludov and Wolffe (2000) Nuc Acids Res 28:1921-1928, and references cited therein). Methyltransferases are required for de novo methylation that occurs in the genome following embryo implantation and for the de novo methylation of newly integrated retroviral sequences in mouse ES cells (Okano et al. (1999) Cell 99:247-257). Proteins having significant homology to vertebrate methyltransferases been identified in zebrafish, Arabidopsis thaliana and maize (Okano et al. (1998) Nature Genet 19:219-220; Cao et al. (2000) PNAS USA 97:4979-4984).
In addition to the methyltransferases, a group of proteins which bind to methylated CpG sequences have also been identified. The methyl-CpG-binding protein MECP2 has been most characterized. MECP2 has been shown to selectively reocgnize methylated DNA and to repress transcription in methylated regions of the genome (Lewis et al. (1992) Cell 69:905-914). MECP2 contains at least two domains: the methyl-CpG-binding domain (MBD), which recognizes symmetrically methylated CpG dinucleotides through contacts in the major groove of the double helix (Wakefield et al. (1999) J. Mol. Biol. 291:1055-1065) and a transcriptional repression domain (TRD), which interacts with several other regulatory proteins (Nan et al. (1997) Cell 88:471-481. MECP2 selectively represses transcription of methylated templates in the absence of an organized chromatin structure and, when tethered to a specific heterologous Gal4-binding domain, its TRD confers transcriptional repression by interacting with TFIIB, a component of the basal transcription machinery (Kaludov et and Wolffe, (2000) Nucleic Acids Res. 28:1921-1928). Methyl binding domain proteins associate with corepressor complexes that include histone deacetylases. Methyl CpG binding proteins have also been shown to be components of chromatin-remodeling complexes, for example the MECP2 repressor complex. Recruitment of a histone deacetylase occurs indirectly through its interaction with the Sin3A adaptor proteins, which causes transcriptional silencing, in part by deacetylation of histones, directing the formation of stable repressive chromatin structures.
Thus, methylation of DNA can repress transcription through multiple mechanisms (see, e.g., Kaludov and Wolffe (2000) Nuc Acids Res 28:1921-1928, and references cited therein). Pathways of repression include direct inhibition of transcription through the failure of transcription factors to associate with methylated recognition elements (Iguchi-Arigan et al. (1989) Genes Dev. 3:612-619) and indirect pathways involving either occlusion of methylated sequences by transcriptional repressors that recognize methylated DNA (Meehan et al. (1992) Nucleic Acids Res. 20:5085-5092) or the modification of chromatin structure targeted by methyl-CpG-specific transcriptional repressors (Buschhausen et al. (1987) PNAS USA 84:1177-1181; Kass et al. (1997) Curr. Biol. 7:157-165).
Despite the characterization of the functional properties of methyl-CpG-specific binding proteins and their constituent MBDs, it has not heretofore been possible to target the various functional activities of MBDs, for use in specific and directed modulation of gene expression.